Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli -: Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose

被引:92
作者
Breazeale, SD
Ribeiro, AA
Raetz, CRH [1 ]
机构
[1] Duke Univ, Med Ctr, Dept Biochem, Durham, NC 27710 USA
[2] Duke Univ, Med Ctr, Duke NMR Spect Ctr, Durham, NC 27710 USA
[3] Duke Univ, Med Ctr, Dept Radiol, Durham, NC 27710 USA
关键词
D O I
10.1074/jbc.M109377200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Addition of the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system. The sequences of the products of the Salmonella typhimurium pmrE andpmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for L-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R. J., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 18503-18514). We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of [alpha-P-32]UDP-glucuronic acid, followed by transamination to generate [alpha-P-32]UDP-L-Ara4N, when NAD and glutamate are added as co-substrates. In addition, the [alpha-(32)p]UDP-L-Ara4N is formylated when N-10-formyltetrahydrofolate is included. These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon. PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-L-threo-pentapyranosyl-4"-ulose diphosphate). A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone. ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.
引用
收藏
页码:2886 / 2896
页数:11
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